The present invention relates to crude drug extracts containing soluble silicon compounds as an effective component, methods for making the crude drug extracts, and also to standardization methods for crude drugs and their extracts.
Living organisms conducting living phenomenon are composed of cells where functional abnormality in the cells introduces the living organisms to a diseased state. Living organisms survive as an individual by adjusting and maintaining their physical and chemical states to and within certain stable physiological conditions corresponding to changes in internal and external circumstances. It is well known that the maintenance and the normalization of the biofunction are especially carried out by various receptors on cell surfaces and the ion channels such as sodium, potassium, calcium, etc. However, if the above-mentioned biofunctions are unbalanced for some reason and it becomes chronic, so-called morbidity results causing various diseases.
The cell membrane consists of lipid bilayers and has an important and complicated function for maintenance of life such as selective permeability, active transport, generation of bioelectricity, expression of immunoactivity, etc. Although normal cells have fluidity and exhibit self-repairing ability to injury, the fluidity of the cell membrane decreases due to internal and external invasions. Exemplary of such invasions are aging, various diseases, and excessive stress stimulation including viral and bacterial infection. The invasions and consequent decrease in fluidity deteriorates maintenance of the homeostasis of a living body. For example, it is well known that vascular endothelial cells and nerve cells are injured by hyperlipemia, hypertension, diabetes mellitus, aging, smoking, etc. resulting in arteriosclerosis, renal diseases, peripheral nervous disorder, etc.
A known mechanism for adjusting the complicated functions in vivo, is an enzymatic system called the kallikrein-kinin system. With respect to this plasma kallikrein-kinin system, it is believed that a blood coagulation factor XII (a Hageman factor, abbreviated as FXII) is activated due to stimulation by a lesion or an invasion to the tissues in vivo whereby a series of enzymatic reactions is induced. Thus, the activated blood coagulation factor XII (abbreviated as FXIIa) acts on plasma prekallikrein which exists in the same plasma to convert it to plasma kallikrein which is an enzyme in activated form. Then, the plasma kallikrein acts on high-molecular-weight kininogen (abbreviated as HK) in the plasma to liberate bradykinin.
The bradykinin which is a product of the plasma kallikrein-kinin system exhibits various physiological activities such as dilation of peripheral blood vessels, acceleration of permeation of blood vessels, induction of pain, generation of inflammation, migration of leucocytes, etc. Bradykinin has also been known as a mediator for induction of pain, inflammation and allergic reactions. Accordingly, when an excessive liberation and production of bradykinin is inhibited, it is possible to relieve pain, inflammation, allergic syndromes, etc. and to make such unhealthy states normal.
The plasma kallikrein-kinin system acts in vivo having a close relationship with various other enzymatic reaction systems such as the renin-angiotensin system, the blood clotting system, the fibrinolysis system, the complement system as well as the catecholamine and arachidoic acid cascade mainly related to prostaglandins, leukotrienes and thromboxanes. Accordingly, the kallikrein-kinin system is closely related to blood pressure regulating action, action through the blood clotting-fibrinolysis-complement system or bioregulation and improving action for peripheral circulation by various physiologically active substances produced by the arachidoic acid cascade and plays an important role in the regulation of functions in vivo. Thus, the plasma kinin-kallikrein system basically relates to biofunctions and participates in various bioregulation systems. Therefore, it has been suggested that a substance having an effect on the plasma kinin-kallikrein system shows various pharmacological activities.
The present inventor has conducted a study, where a plasma kallikrein-kinin system is utilized, paying attention to silicon compounds which regulate immune action and autonomic action of nerve cells in living organisms. Silicon is widely distributed in the animal and vegetable kingdoms. Especially in animal tissues, it is abundantly present as silicic acid in skin, hair, bone and various organs such as the lung, adrenal gland, thymus, pancreas and spleen. Silicon is known to be an essential component for the formation of bones. Further, in animal tissues, silicic acid forms a collagen cross-linking chain and is contained as a constituting component of acidic mucopolysaccharides and it has been suggested that elasticity of skin is related to the amount of silicic acid.
Crude drugs have been used in medical treatment since ancient time but their quality has been mostly confirmed only by means of empirical extracting methods for many years. For example, in the methods of evaluating the quality of crude drugs in the Japanese Pharmacopoeia (13th Edition), many of the methods are merely a confirmation test by means of color reaction or spots in thin layer chromatography. Under such circumstances, there has been a strong demand for establishing a standardization for the substances so that a predetermined effect for the crude drug can be guaranteed. Standardization for quality of various crude drugs by means of a substantial index related to pharmaceutical effect would contribute to providing crude drug extracts having a stable quality and would greatly contribute to an appropriate standardization of pharmaceuticals.
The present inventor has studied silicon compounds in living organisms and regulated the quality of crude drugs using silicon compounds to express their pharmaceutical effect in vivo whereupon the present invention has been accomplished. The present invention provides crude drug extracts containing soluble silicon compounds as an effective component which exhibit inhibitory action against the production of plasma kallikrein. The present invention also provides methods for manufacturing crude drug extracts, and standardizing methods for crude drugs and extracts thereof.
A crude drug extract which inhibits production of plasma kallikrein may be produced by subjecting a plant or animal crude drug to extraction with water or an aqueous-based extracting solvent to obtain an extract having at least about 0.05 mg of at least one soluble silicon compound calculated as silicon per gram of dry extract, as an effective component. In preferred embodiments, an alkaline extracting solvent is employed to substantially increase the soluble silicon compound content of the crude drug extract. The extracting solvent may have a pH of from about 8.5 to about 10.5. The reaction may be conducted at room temperature, or the extract may be heated or boiled, followed by evaporating the solvent therefrom to give a dry substance. Examples of aqueous-based extraction solvents include mixtures of water with ethanol or phenol. In embodiments of the invention, prior to extraction with the alkaline extracting solvent, the crude drug may be subjected to extraction with an extracting solvent having a neutral pH. The crude drug extracts of the present invention may be used in pharmaceutically effective amounts as an anti-allergic agent, a sedative, an analgesic agent, or an anti-inflammatory agent to treat patients in need thereof.
Standardizing or evaluating animal or plant crude drugs for pharmaceutical effectiveness may be achieved by subjecting an animal or plant crude drug to extraction to obtain an extract solution, drying the extract solution to obtain a dry crude drug extract, determining the amount of soluble silicon compounds calculated as silicon per gram of dry crude drug extract, and comparing the determined amount of soluble silicon compounds to a minimal amount needed to obtain inhibition of plasma kallikrein production. In embodiments of the invention, standardizing crude drug extracts, such as plant crude drug extracts may be performed by determining the amount of soluble silicon compounds calculated as silicon per gram of dry crude drug extract, measuring inhibiting action of the crude drug extract against plasma kallikrein, and subjecting the crude drug extract to a plurality of coloring reactions.
The present invention relates to crude drug extracts which contain not less than 0.05 mg of soluble silicon compounds calculated as silicon per gram of the dry crude drug. The crude drug extract of the present invention may be obtained by extraction of various crude drugs, for example, animal and plant crude drugs including tanjin (Salviae militiorrhizae radix), shireishi (Ganoderma lucidum), creeping saxifrage (Saxifraga stolonifera), scouring rush (Equisetum hiemale), Chinese gutta percha, plantago herb, plantago seeds, chorei (polyporus sclerotium), saiko (bupleurum root), Japanese angelica root, elderberry, bukuryo (poria sclerotium), pueraria root, crude aloe, ginseng, ginger, alisma rhizome, schisandra fruit, sanshiti (root of Panax nothoginseng), jujube, chinpi (citrus unshiu peel), bakumondo (ophiopogon tuber), young staghorn, oriental bezoar, lumbicusa, bear bile, longgu, etc. The present invention is applicable to any crude drug extract derived from animals, plants, minerals, etc. satisfying the requirements of the present invention. Such crude drug materials are extracted with water, ethanol or a suitable extracting solvent to which an additive such as phenol is added whereby the crude drug extract can be manufactured. At that time, extraction and concentration of the active substances may be enhanced by heating or changing the pH of the solvent. Thus, the following manufacturing methods may be exemplified for obtaining crude drug extracts in accordance with present invention:
1) Pure water is added to a crude drug material, the mixture is boiled with stirring and the insoluble matters are removed by filtration or the like to give an extract solution. The extract solution is concentrated if necessary and spray-dried or freeze-dried in vacuo to give a powder.
2) Pure water is added to a crude drug material, the mixture is boiled with stirring and the insoluble matters are removed by filtration or the like to obtain an extract. Pure water is further added to the residue, pH is adjusted to an alkaline region (to 8.5-10.5, for example to around 9.5), then the mixture is boiled with stirring again, and the insoluble matters are removed by filtration or the like to obtain an extract, and the extract is adjusted to about the neutral pH region and combined with the already-prepared first extract. Then, the combined extract is concentrated and/or evaporated to dryness if necessary to give a powder. Spray-drying or freeze-drying in vacuo to give a powder may be employed as in the above-mentioned method 1).
3) To a crude drug material is added a 1% aqueous phenol solution followed by boiling with stirring and the insoluble matters are removed by filtration or the like to give an extract solution. The extract solution is concentrated if necessary and spray-dried or freeze-dried in vacuo to give a powder.
4) Pure water and ethanol are added to a crude drug material, the mixture is boiled with stirring and the insoluble matters are removed by filtration or the like to give an extract solution. The extract solution is concentrated if necessary and spray-dried or freeze-dried in vacuo to give a powder.
5) After conducting the extracting operations as described in the above methods 1) to 4), the pH of the extract is adjusted to weakly alkaline (for example, to pH of around 8.5) followed by concentrating, and the pH of the concentrate is adjusted to nearly the neutral region followed by pulverizing in the same manner as mentioned above.
Conventional pH adjusting agents, such as inorganic or organic bases and acids and salts may be employed to obtain a desired pH for the extracting solvent and extract. For example, alkali metal hydroxides such as sodium hydroxide, and potassium hydroxide, etc. may be employed to obtain a desired alkaline pH. Exemplary acids which may be employed to adjust pH to the neutral range include hydrochloric acid, sulfuric acid, and hydrobromic acid, etc.
In accordance with the present invention, crude drug extracts are characterized and evaluated for pharmaceutical effectiveness by specifying the soluble silicon compound content of the crude drug extract. The content of the soluble silicon compounds in the dried crude drug extract obtained by the above-mentioned manufacturing methods can be analyzed by the following method and is able to be regulated as an amount calculated as silicon.
Thus, the crude drug extract is added to water (to an extent of 1 mg/ml), the mixture is subjected to stirring and an ultrasonic treatment. In preferred embodiments, the stirring is conducted at room temperature for about ten minutes and ultrasonic treatment is conducted at room temperature for about ten minutes. Then, the insoluble matters are removed by filtration or centrifugation, and the amount of silicon in the resulting solution is measured by a molybdenum blue method. In addition, an inhibiting action of the same sample solution against the production of plasma kallikrein is measured and is confirmed as an index for the soluble silicon compounds. The measured inhibiting action against plasma kallikrein production is important as an index for the measurement and confirmation of the titer (potency of the biological activity) of the soluble silicon compounds having a biological activity.
In addition to the crude drug extract in which the soluble silicon compounds are specified, the present invention further covers various embodiments such as a method of manufacturing the same and its pharmaceutical use. Preferred embodiments of the present invention are:
(1) A crude drug extract containing 0.05 mg or more soluble silicon compounds (calculated as silicon) per gram of the dry substance.
(2) A crude drug extract according to the above paragraph (1) which is extracted from one of the animal and plant crude drugs selected from the group consisting of tanjin (Salvia militiorrhiza radix), shireishi (Ganoderma lucidum), creeping saxifrage (Saxifraga stolonifera), scouring rush (Equisetum hiemale), Chinese gutta percha, plantago herb, plantago seeds, chorei (polyporus sclerotium), saiko (bupleurum root), Japanese angelica root, elderberry, bukuryo (poria sclerotium), pueraria root, crude aloe, ginseng, ginger, alisma rhizome, schisandra fruit, sanshiti (root of Panax nothoginseng), jujube, chinpi (citrus unshiu peel), bakumondo (ophiopogon tuber), young staghorn, oriental bezoar, lumbicusa, bear bile, longgu, etc.
(3) A crude drug extract according to the above paragraph (1) which is extracted from one of the crude drugs selected from the group consisting of tanjin (Salvia militiorrhiza radix), shireishi (Ganoderma lucidum), creeping saxifrage (Saxifraga stolonifera), scouring rush (Equisetum hiemale), Chinese gutta percha, plantago herb, plantago seeds, chorei (polyporus sclerotium), saiko (bupleurum root), Japanese angelica root, elderberry, bukuryo (poria sclerotium), pueraria root, crude aloe and ginseng.
(4) A crude extract according to the above paragraph (1), (2) or (3) in which water is added to the dry substance (to an extent of 1 mg/mL) and the amount of the silicon compounds existing in a solubilized state in said aqueous solution is regulated or specified.
(5) A method for the manufacture of a crude drug extract according to any one of the above paragraphs (1) to (4) in which an extracting solvent where the pH is adjusted to an alkaline region is used.
(6) A manufacturing method according to the above paragraph (5) in which an extracting solvent where the pH is adjusted to from 8.5 to 10.5 is used.
(7) A method for the manufacture of a crude drug extract according to any one of the above paragraphs (1) to (4) in which an extraction is conducted using an extracting solvent near the neutral pH region (pH of about 7), followed by using an extracting solvent where the pH is adjusted to an alkaline region, such as a pH of from 8.5 to 10.5.
(8) A manufacturing method according to any of the above paragraphs (5) to (7) in which the crude drug material is extracted by heating or boiling followed by evaporating the solvent therefrom to give a dry substance.
(9) A manufacturing method according to any of the above paragraphs (5) to (8) in which water, ethanol or a mixed solution thereof is used as an extracting solvent.
(10) A manufacturing method according to the above paragraph (9) in which an extracting solvent to which an additive such as phenol is added is employed.
(11) A method for the standardization of a crude drug extract in which soluble silicon compounds are used as an index.
(12) A method according to the above paragraph (9) in which a crude drug mentioned in paragraph (2) or (3) is standardized.
(13) A method according to the above paragraph (12) in which the crude drug is standardized by a method according to paragraph (4) above.
(14) A method according to any of the above paragraphs (11) to (13) in which the crude drug is standardized by combining identifying tests such as a coloring reaction.
(15) An inhibiting agent against plasma kallikrein production containing a crude extract according to any of paragraphs (1) to (4) above as an effective component.
(16) An inhibiting agent against plasma kallikrein production according to the above paragraph (15) in which said agent is an anti-allergic agent.
(17) An inhibiting agent against plasma kallikrein production according to the above paragraph (15) in which said agent is a sedative or an analgesic agent.
(18) An inhibiting agent against plasma kallikrein production according to the above paragraph (15) in which said agent is an anti-inflammatory agent.
An extract of a crude drug of the present invention can be used as a drug material in extract form or a dried powder form. The crude drug extract can be made into pharmaceutical preparations as is without any excipients or together with commonly-used excipients. For example, a crude drug extract which is an effective component of the pharmaceutical compositions of the present invention can be made into various pharmaceutical compositions or preparations by combining one or more of the extracts with at least one pharmaceutical carrier or diluent. The extracts can be made into various types of preparations by known methods. The pharmaceutical preparations or compositions may be made into solid, semi-solid, liquid or aerosol formulations for oral administration (e.g. tablets, capsules, powders, liquids, etc.) and for parenteral administration (e.g. for subcutaneous, intravenous, intramuscular, intrarectal and intranasal administrations).
The extracts of the present invention may be used either solely or jointly in pharmaceutically effective amounts for treating animals or humans. They may also be used in pharmaceutically effective amounts in combination with pharmaceutically effective amounts of other pharmaceutically active components in pharmaceutical compositions or preparations.
In the case of preparations for oral administration, one or more of the extracts of the present invention alone or together with commonly-used pharmaceutically acceptable excipients in pharmaceutically acceptable amounts. For example, for oral administration, the extract can be used as a pharmaceutical preparation preferably as is or together with buffers, preservatives, flavors such as saccharides, perfumes, etc. When making the extract dry and into a powder form, the dried powder as is or together with commonly-used excipients such as a suitable pharmaceutically acceptable additive or carrier (e.g. lactose, mannitol, corn starch, potato starch, etc.) may be mixed with one or more pharmaceutically acceptable: (1) binders such as crystalline cellulose, cellulose, cellulose derivatives, gum arabicum, corn starch, gelatin, etc., (2) disintegrating agents such as corn starch, potato starch, potassium carboxymethyl-cellulose, etc., (3) lubricating agents such as talc, magnesium stearate, etc., and (4) other pharmaceutically acceptable excipients including pharmaceutically acceptable bulking agents, moisturizing agents, buffers, preservatives, perfumes and the like to obtain tablets, diluted powders, granules or capsules.
In the case of parenteral administration using injections, for example, it is possible to prepare solutions or suspensions of one or more extracts of the present invention in pharmaceutically acceptable carriers such as aqueous and nonaqueous solvents such as distilled water for injection, physiological saline solution, Ringer""s solution, plant oil, synthetic fatty acid glycerides, higher fatty acid esters, propylene glycol, etc.
It is also possible, depending upon the type of the disease, to prepare pharmaceutical preparations other than the above-mentioned ones such as suppositories, inhalations, aerosol preparations, collyriums, ointments, poultices, etc.
For example, suppositories may be prepared by mixing at least one extract of the present invention with pharmaceutically acceptable amounts of one or more pharmaceutically acceptable fatty/oily bases (e.g. cacao butter), emulsified bases, water-soluble bases (e.g. Macrogol), hydrophilic bases, etc.
In the case of inhalations or aerosol preparations, at least one extract of the present invention in the form of a liquid or minute powder can be filled up in an aerosol container with a gas or liquid spraying agent, and if desired, with conventional adjuvants such as one or more pharmaceutically acceptable humidifying agents or dispersing agents. They can also be used as pharmaceuticals for a non-pressurized preparation such as in a nebulizer or an atomizer.
In order to make the extracts of the present invention into collyriums, they can be prepared as a solution or suspension together with an aqueous solvent such as sterile, purified water and physiologically saline solution, or a non-aqueous solvent for injection. The collyriums may also include pharmaceutically acceptable preservants, sterilizing agents, pH adjusting agents, and the like.
The present invention will now be further illustrated by way of the following non-limiting examples wherein all parts, percentages, and ratios are by weight, all temperatures are in xc2x0 C. or room temperature, and all pressures are in psia or atmospheric unless otherwise indicated: